The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Mutator transposase is widespread in the grasses

Although the Mutator (Mu) system is well characterized in maize (Zea mays), very little is known about this highly mutagenic system of transposons in other grasses. Mutator is regulated by the MuDR class of elements, which encodes two genes, one of which, mudrA, has similarity to a number of bacterial transposases. Experiments in our laboratory, as well as database searches, demonstrate that mudrA sequences are ubiquitous and diverse in the grasses. In several species it is clear that multiple paralogous elements can be present in a single genome. In some species such as wheat (Triticum aestivum) and rice (Oryza sativa), mudrA-similar sequences are represented in cDNA databases, suggesting the presence of active Mu transposon systems in these species. Further, in rice and in sorghum, mudrA-like genes are flanked by long terminal inverted repeats, as well as the short host sequence direct repeats diagnostic of insertion. Thus, there is ample evidence that systems related to Mu in maize are at least potentially active in a wide variety of grasses. However, the mudrB gene, though important for Mu activity in maize, is not necessarily a component of Mu elements in other grasses.     

Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.     

Sequence similarity of putative transposases links the maize Mutator autonomous element and a group of bacterial insertion sequences.

The Mutator transposable element system of maize is the most active transposable element system characterized in higher plants. While Mutator has been used to generate and tag thousands of new maize mutants, the mechanism and regulation of its transposition are poorly understood. The Mutator autonomous element, MuDR, encodes two proteins: MURA and MURB. We have detected an amino acid sequence motif shared by MURA and the putative transposases of a group of bacterial insertion sequences. Based on this similarity we believe that MURA is the transposase of the Mutator system. In addition we have detected two rice cDNAs in genbank with extensive similarity to MURA. This sequence similarity suggests that a Mutator-like element is present in rice. We believe that Mutator, a group of bacterial insertion sequences, and an uncharacterized rice transposon represent members of a family of transposable elements.